Pharmaceutical compositions comprising troponin subunits, fragments and analogs thereof and methods of their use to inhibit angiogenesis

ABSTRACT

The present invention relates to pharmaceutical compositions containing troponin subunits C, I, or T, or fragments thereof, in therapeutically effective amounts that are capable of inhibiting endothelial cell proliferation. The invention also relates to pharmaceutical compositions containing analogs of troponin subunits C, I, or T and analogs of their fragments, in therapeutically effective amounts that are capable of inhibiting endothelial cell proliferation. The invention further relates to treatment of neovascular disorders by administration of a therapeutic compound of the invention.

The present invention provides for a novel pharmaceutical composition,and method of use thereof for the treatment of diseases or disordersinvolving abnormal angiogenesis.

More particularly, the present invention is based, in part, on thediscovery that troponin subunits C, I and T inhibit stimulatedendothelial cell proliferation. Pharmaceutical compositions containingtherapeutically effective amounts of troponin C, I, or T, subunits,fragments, or analogs and methods of therapeutic use thereof areprovided.

BACKGROUND

Angiogenesis, the process of new blood vessel development and formation,plays an important role in numerous physiological events, both normaland pathological. Angiogenesis occurs in response to specific signalsand involves a complex process characterized by infiltration of thebasal lamina by vascular endothelial cells in response to angiogenicgrowth signal(s), migration of the endothelial cells toward the sourceof the signal(s), and subsequent proliferation and formation of thecapillary tube. Blood flow through the newly formed capillary isinitiated after the endothelial cells come into contact and connect witha preexisting capillary.

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., 1989, Cell 56:345-355. In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail.

Unregulated angiogenesis becomes pathologic and sustains progression ofmany neoplastic and non-neoplastic diseases. A number of seriousdiseases are dominated by abnormal neovascularization including solidtumor growth and metastases, arthritis, some types of eye disorders, andpsoriasis. See, e.g., reviews by Moses et al., 1991, Biotech. 9:630-634;Folkman et al., 1995, N. Engl. J. Med., 333:1757-1763; Auerbach et al.,1985, J. Microvasc. Res. 29:401-411; Folkman, 1985, Advances in CancerResearch, eds. Klein and Weinhouse, Academic Press, New York, pp.175-203; Patz, 1982, Am. J. Opthalmol. 94:715-743; and Folkman et al.,1983, Science 221:719-725. In a number of pathological conditions, theprocess of angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, 1987,Science 235:442-447.

The maintenance of the avascularity of the cornea, lens, and trabecularmeshwork is crucial for vision as well as to ocular physiology. Thereare several eye diseases, many of which lead to blindness, in whichocular neovascularization occurs in response to the diseased state.These ocular disorders include diabetic retinopathy, neovascularglaucoma, inflammatory diseases and ocular tumors (e.g.,retinoblastoma). There are also a number of other eye diseases which arealso associated with neovascularization, including retrolentalfibroplasia, uveitis, retinopathy of prematurity, macular degeneration,and approximately twenty eye diseases which are associated withchoroidal neovascularization and approximately forty eye diseasesassociated with iris neovascularization. See, e.g., reviews by Waltmanet al., 1978, Am. J. Ophthal. 85:704-710 and Gartner et al., 1978, Surv.Ophthal. 22:291-312. Currently, the treatment of these diseases,especially once neovascularization has occurred, is inadequate andblindness often results. Studies have suggested that vaso-inhibitoryfactors which are present in normal ocular tissue (cornea and vitreous)are lost in the diseased state.

An inhibitor of angiogenesis could have an important therapeutic role inlimiting the contributions of this process to pathological progressionof the underlying disease states as well as providing a valuable meansof studying their etiology. For example, agents that inhibit tumorneovascularization could play an important role in inhibiting metastatictumor growth.

The components of angiogenesis relating to vascular endothelial cellproliferation, migration and invasion, have been found to be regulatedin part by polypeptide growth factors. Experiments in culture, indicatethat endothelial cells exposed to a medium containing suitable growthfactors can be induced to evoke some or all of the angiogenic responses.Several polypeptides with in vitro endothelial growth promoting activityhave been identified. Examples include acidic and basic fibroblastgrowth factors, transforming growth factors α and β, platelet-derivedendothelial cell growth factor, granulocyte colony-stimulating factor,interleukin-8, hepatocyte growth factor, proliferin, vascularendothelial growth factor and placental growth factor. See, e.g., reviewby Folkman et al., 1995, N. Engl. J. Med., 333:1757-1763.

Although extracts from several different tissue sources have been shownto contain anti-angiogenic activity, several molecules such as plateletfactor-4, thrombospondin, protamine, and transforming growth factor B,have been found to negatively regulate different aspects ofangiogenesis, such as cell proliferation or cell migration, no singletissue-derived macromolecule capable of inhibiting angiogenesis has beenidentified in the prior art. See, e.g., reviews by Folkman, J., 1995, N.Engl. J. Med. 333:1757-1763 and D'Amore, 1985, Prog. Clin. Biol. Res.221:269-283. There is therefore a great need for the furtheridentification and characterization of chemical agents which can preventthe continued deregulated spread of vascularization and which wouldpotentially have broad applicability as a therapy for those diseases inwhich neovascularization plays a prominent role.

Capillary endothelial cells ("EC") proliferate in response to anangiogenic stimulus during neovascularization. Ausprunk and Folkman,1977, J. Microvasc. Res. 14:153-65. An in vitro assay assessingendothelial cell proliferation in response to known angiogenesissimulating factors, such as acidic or basic fibroblast growth factor(aFGF and bFGF, respectively), has been developed to mimic the processof neovascularization in vitro. This type of assay is the assay ofchoice to demonstrate the stimulation of capillary EC proliferation byvarious angiogenic factors. Shing et al., 1984, Science 223:1296-1298.

The process of capillary EC migration through the extracellular matrixtowards an angiogenic stimulus is also a critical event required forangiogenesis. See, e.g., review by Ausprunk et al., 1977, J. Microvasc.Res. 14:53-65. This process provides an additional assay by which tomimic the process of neovascularization in vitro. A modification of theBoyden chamber technique has been developed to monitor EC migration.Boyden et al., 1962, J. Exptl. Med. 115:453-456, Example 4. To date,only a few tissue-derived EC cell migration inhibitors are known. See,e.g., review by Langer et al., 1976, Science 193:70-72.

In the early 1970's, a number of in vivo angiogenesis model bioassayswere widely used. These model systems included rabbit corneal pocket,chick chorioallantoic membrane ("CAM"), rat dorsal air sac and rabbitair chamber bioassays. For review, see, Blood et al., 1990, Biochem. etBiophys. Acta 1032:89-118. The development of controlled releasepolymers capable of releasing large molecules such as angiogenesisstimulators and inhibitors was critical to the use of these assays.Langer et al., 1976, Nature 263:797-800.

In the CAM bioassay, fertilized chick embryos are cultured in Petridishes On day 6 of development, a disc of a release polymer, such asmethyl cellulose, impregnated with the test sample or an appropriatecontrol substance is placed onto the vascular membrane at its advancingedge. On day 8 of development, the area around the implant is observedand evaluated. Avascular zones surrounding the test implant indicate thepresence of an inhibitor of embryonic neovascularization. Moses et al.,1990, Science, 248:1408-1410 and Taylor et al., 1982, Nature,297:307-312. The reported doses for previously described angiogenesisinhibitors tested alone in the CAM assay are 50 μg of protamine (Tayloret al. (1982)), 200 μg of bovine vitreous extract (Lutty et al., 1983,Invest. Opthalmol. Vis. Sci. 24:53-56), and 10 μg of platelet factor IV(Taylor et al. (1982)). The lowest reported doses of angiogenesisinhibitors effective as combinations include heparin (50 μg) andhydrocortisone (60 μg), and B-cyclodextrin tetradecasulfate (14 μg) andhydrocortisone (60 μg), reported by Folkman et al., 1989, Science243:1490.

According to the rabbit corneal pocket assay, polymer pellets ofethylene vinyl acetate copolymer ("EVAC") are impregnated with testsubstance and surgically implanted in a pocket in the rabbit corneaapproximately 1 mm from the limbus. Langer et al., 1976, Science193:707-72. To test for an angiogenesis inhibitor, either a piece ofcarcinoma or some other angiogenic stimulant is implanted distal to thepolymer 2 mm from the limbus. In the opposite eye of each rabbit,control polymer pellets that are empty are implanted next to anangiogenic stimulant in the same way. In these control corneas,capillary blood vessels start growing towards the tumor implant in 5-6days, eventually sweeping over the blank polymer. In test corneas, thedirectional growth of new capillaries from the limbal blood vesseltowards the tumor occurs at a reduced rate and is often inhibited suchthat an avascular region around the polymer is observed. This assay isquantitated by measurement of the maximum vessel lengths with astereospecific microscope.

Troponin, a complex of three polypeptides is an accessory protein thatis closely associated with actin filaments in vertebrate muscle. Thetroponin complex, acts in conjunction with the muscle form oftropomyosin to mediate the Ca²⁺ dependency of myosin ATPase activity andthereby regulate muscle contraction. The troponin polypeptides T, I, andC, are named for their tropomyosin binding, inhibitory, and calciumbinding activities, respectively. Troponin T binds to tropomyosin and isbelieved to be responsible for positioning the troponin complex on themuscle thin filament. Troponin I binds to actin, and the complex formedby troponins I and T, and tropomyosin, inhibits the interaction of actinand myosin. Troponin C is capable of binding up to four calciummolecules. Studies suggest that when the level of calcium in the muscleis raised, troponin C causes troponin I to loose its hold on the actinmolecule, causing the tropomyosin molecule shift, thereby exposing themyosin binding sites on actin and stimulating myosin ATPase activity.Prior to the discovery of the present invention, troponin subunits werenot known to inhibit the process of endothelial cel proliferation.

The citation of a reference herein shall not be construed as anadmission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions containingtroponin subunits C, I, or T, or fragments thereof, in therapeuticallyeffective amounts that are capable of inhibiting endothelial cellproliferation. The invention also relates to pharmaceutical compositionscontaining analogs of troponin subunits C, I, or T and analogs of theirfragments, in therapeutically effective amounts that are capable ofinhibiting endothelial cell proliferation. The invention further relatesto treatment of neovascular disorders by administration of a therapeuticcompound of the invention. Such therapeutic compounds (termed herein"Therapeutics"), include: troponin subunits C, I, and T, and fragmentsand analogs thereof. In one embodiment, a Therapeutic of the inventionis administered to treat a cancerous condition, or to preventprogression from the pre-neoplastic or pre-malignant state into aneoplastic or a malignant state. In other specific embodiments, aTherapeutic of the invention is administered to treat ocular disordersassociated with neovascularization.

Definitions

As used herein,:

The term "troponin subunit", when not preceding the terms C, I or T,means generically any of troponin subunits C, I, or T.

BRIEF DESCRIPTION OF THE FIGURES.

FIG. 1. Inhibition of bovine capillary Endothelial Cell (BCE)proliferation by troponin C. Percent inhibition of bFGF-stimulated BCEproliferation is shown as a function of troponin C concentration (nM).Percent inhibition was determined by comparing results obtained forcells treated with stimulus alone with those obtained for samplesexposed to both stimulus and inhibitor. Well volume was 200 μl.

FIG. 2. Inhibition of capillary BCE proliferation by troponin I. Percentinhibition of bFGF-stimulated BCE proliferation is shown as a functionof troponin I concentration (nM). Percent inhibition was determined asdescribed in FIG. 1. Well volume was 200 μl.

FIG. 3. Inhibition of capillary BCE proliferation by troponin T. Percentinhibition of bFGF-stimulated BCE proliferation is shown as a functionof troponin T concentration (nM). Percent inhibition was determined asdescribed in FIG. 1. Well volume was 200 μl.

FIG. 4. Inhibition of BCE proliferation by troponins C and I. Percentinhibition of bFGF-stimulated BCE proliferation is shown as a functionof troponin I and C concentration (nM). Percent inhibition wasdetermined as described in FIG. 1. Well volume was 200 μl.

FIG. 5. Inhibition of capillary BCE proliferation by troponin C, I andT. Percent inhibition of bFGF-stimulated BCE proliferation is shown as afunction of troponin C, I, and T concentration (nM). Percent inhibitionwas determined as described in FIG. 1. Well volume was 200 μl.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to therapeutic methods and compositionsbased on troponin subunits. The invention provides for treatment ofneovascular disorders by administration of a therapeutic compound of theinvention. Such therapeutic compounds (termed herein "Therapeutics")include: troponin C, I, and T subunits, fragments and analogs thereof(collectively "peptides of the invention"). The peptides of theinvention are characterized by the property of inhibiting bovineendothelial cell proliferation in culture with an IC₅₀ of 10 μM or less.In a preferred embodiment, a Therapeutic of the invention isadministered to treat a cancerous condition, or to prevent progressionfrom a pre-neoplastic or non-malignant state into a neoplastic or amalignant state. In other specific embodiments, a Therapeutic of theinvention is administered to treat an ocular disorder associated withneovascularization.

In a preferred aspect, a Therapeutic of the invention is a peptideconsisting of at least a fragment of troponin C, troponin I, troponin T,or troponins C and I, which is effective to inhibit endothelial cellproliferation.

Examples of the troponin subunits that can be utilized in accordancewith the invention, include the subunits of troponin from human fasttwitch skeletal muscle, the sequences of which are given below:

    __________________________________________________________________________    Human Fast Twitch Skeletal Muscle Troponin C (SEQ ID NO:1)                     1 M T D Q Q A E A R S Y L S E E M I A E F                                     21                                                                              K A A F D M F D A D G G G D I S V K    E                                                                        L                                         41                                                                              G T V M R M L G Q T P T K E E L D A    I                                                                        I                                         61                                                                              E E V D E D G S G T I D F E E F L V    M                                                                        M                                         81                                                                              V R Q M K E D A K G K S E E E L A E    C                                                                        F                                        101                                                                              R I F D R N A D G Y I D P E E L A E    I                                                                        F                                        121                                                                              R A S G E H V T D E E I E S L M K D    G                                                                        D                                        141                                                                              K N N D G R I D F D E F L K M M E G    V                                                                        Q                                        Human Fast Twitch Skeletal Muscle Troponin I (SEQ ID NO:2)                     1 M G D E E K R N R A I T A R R Q H L K S                                     21                                                                              V M L Q I A A T E L E K E E S R R E    A                                                                        E                                         41                                                                              K Q N Y L A E H C P P L H I P G S M    S                                                                        E                                         61                                                                              V Q E L C K Q L H A K I D A A E E E    K                                                                        Y                                         81                                                                              D M E V R V Q K T S K E L E D M N Q    K                                                                        L                                        101                                                                              F D L R G K F K R P P L R R V R M S    A                                                                        D                                        121                                                                              A M L K A L L G S K H K V C M D L R    A                                                                        N                                        141                                                                              L K Q V K K E D T E K E R D L R D V    G                                                                        D                                        161                                                                              W R K N I E E K S G M E G R K K M F    E                                                                        S                                        181                                                                              E S                                                                        Human Fast Skeletal Beta Troponin T (SEQ ID NO:3)                              1 M S D E E V E Q V E E Q Y E E E E E A Q                                     21                                                                              E E E E V Q E D T A E E D A E E E K    P                                                                        R                                         41                                                                              P K L T A P K I P E G E K V D F D D    I                                                                        Q                                         61                                                                              K K R Q N K D L M E L Q A L I D S H    F                                                                        E                                         81                                                                              A R K K E E E E L V A L K E R I E K    R                                                                        R                                        101                                                                              A E R A E Q Q R I R A E K E R E R Q    N                                                                        R                                        121                                                                              L A E E K A R R E E E D A K R R A E    D                                                                        D                                        141                                                                              L K K K K A L S S M G A N Y S S Y L    A                                                                        K                                        161                                                                              A D Q K R G K K Q T A R E M K K K I    L                                                                        A                                        181                                                                              E R R K P L N I D H L G E D K L R D    K                                                                        A                                        201                                                                              K E L W E T L H Q L E I D K F E F G    E                                                                        K                                        221                                                                              L K R Q K Y D I T T L R S R I D Q A    Q                                                                        K                                        241                                                                              H S K K A G T P A K G K V G G R W K                                        __________________________________________________________________________

In another embodiment, the invention encompasses peptides which arehomologous to human fast-twitch skeletal troponin C (SEQ ID NO:1) orfragments thereof. In one embodiment, the amino acid sequence of thepeptide has at least 80% identity compared to the fragment of humanfast-twitch skeletal troponin C from which it is derived (the "prototypefragment"). In another embodiment, this identity is greater than 85%. Ina more preferred embodiment, this identity is greater than 90%. In amost preferred embodiment, the amino acid sequence of the peptide has atleast 95% identity with the prototype fragment. Fragments can be atleast 10 amino acids, and in preferred embodiments at least 50, 75, 100and 120 amino acids, respectively.

In another embodiment, the invention encompasses peptides which arehomologous to human fast-twitch skeletal troponin I(SEQ ID NO:2) orfragments thereof. In one embodiment, the amino acid sequence of thepeptide has at least 80% identity with the prototype human fast-twitchskeletal troponin I fragment. In another embodiment, this identity isgreater than 85%. In a more preferred embodiment, this identity isgreater than 90%. In a most preferred embodiment, the amino acidsequence of the peptide has at least 95% identity with the prototypefragment. Fragments can be at least 10 amino acids, and in preferredembodiments at least 50, 75, 100 and 120 amino acids, respectively.

In another embodiment, the invention encompasses peptides which arehomologous to human fast-twitch skeletal troponin T (SEQ ID NO:3) orfragments thereof. In one embodiment, the amino acid sequence of thepeptide has at least 80% identity with the prototype human fast-twitchskeletal beta troponin T. In another embodiment, this identity isgreater than 85%. In a more preferred embodiment, this identity isgreater than 90%. In a most preferred embodiment, the amino acidsequence of the peptide has at least 95% identity with the prototypefragment. Fragments can be at least 10 amino acids, and in preferredembodiments at least 50, 75, 100, 120 and 200 amino acids in length,respectively.

In other specific embodiments, the peptides of the invention aretroponin C, troponin I and troponin T subunits of the fast twitch, slowtwitch and cardiac isoforms from other mammalian species, e.g., human,rabbit, rat, mouse, bovine, ovine and porcine.

In a specific embodiment, a Therapeutic of the invention is combinedwith a therapeutically effective amount of another molecule whichnegatively regulates angiogenesis which may be, but is not limited to,platelet factor 4, thrombospondin-1, tissue inhibitors ofmetalloproteases (TIMP1 and TIMP2) prolactin (16-Kd fragment),angiostatin (38-Kd fragment of plasminogen), bfGf soluble receptor,transforming growth factor β, interferon alfa, and placentalproliferin-related protein.

Paradoxically, neovascularization gradually reduces a tumorsaccessibility to chemotherapeutic drugs due to increased interstitialpressure within the tumor, which causes vascular compression and centralnecrosis. In vivo results have demonstrated that rodents receivingangiogenic therapy show increased delivery of chemotherapy to a tumor.Teicher et al., 1994, Int. J. Cancer 57:920-925. Thus, in oneembodiment, the invention provides for a pharmaceutical composition ofthe present invention in combination with a chemotherapeutic agent.

In another preferred aspect, a Therapeutic of the invention is combinedwith chemotherapeutic agents or radioactive isotope exposure.

The invention is illustrated by way of examples infra which disclose,inter alia, the inhibition of capillary endothelial cell proliferationby troponin subunits C, I, and T and the means for determininginhibition of capillary endothelial cell migration and inhibition ofneovascularization in vivo by troponin subunits.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections set forthbelow.

TROPONIN SUBUNITS, Fragments AND ANALOGS

The invention provides for pharmaceutical compositions comprisingtroponin subunits, fragments, and analogs thereof. In particularaspects, the subunits, fragments, or analogs are of fly, frog, mouse,rat, rabbit, pig, cow, dog, monkey, or human troponin subunits.

It is envisioned that troponin subunit fragments can be made by alteringtroponin sequences by substitutions, additions or deletions that providefor functionally equivalent molecules. These include, but are notlimited to, troponin subunits, fragments, or analogs containing, as aprimary amino acid sequence, all or part of the amino acid sequence of atroponin subunit including altered sequences in which functionallyequivalent amino acid residues are substituted for residues within thesequence resulting in a silent change. For example, one or more aminoacid residues within the sequence can be substituted by another aminoacid of a similar polarity which acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

One embodiment of the invention provides for molecules consisting of orcomprising a fragment of at least 10 (continuous) amino acids of atroponin subunit which is capable of inhibiting endothelial cellproliferation. In other embodiments, this molecule consists of at least20 or 50 amino acids of the troponin subunit. In specific embodiments,such molecules consist of or comprise fragments of a troponin subunitthat at least 75, 120 or 200 amino acids.

In a preferred embodiment, the protein is a mammalian troponin subunit.In alternative embodiments, it is a mammalian troponin C, I, or Tsubunit.

The troponin subunit fragments and analogs of the invention can bederived from tissue (see, for example, Example 1; Ebashi et al., 1968,J. Biochem. 64:465; Yasui et al., 1968, J. Biol. Chem. 243:735;Hartshorne et al., 1968, Biochem. Biophys. Res. Commun. 31:647; Shaub etal., 1969, Biochem. J. 115:993; Greaser et al., 1971, J. Biol. Chem.246:4226-4733; Brekke et al., 1976, J. Biol. Chem. 251:866-871; andYates et al., 1983, J. Biol. Chem. 258:5770-5774) or produced by variousmethods known in the art. The manipulations which result in theirproduction can occur at the gene or protein level. For example, a clonedtroponin gene sequence coding for troponin subunits C, I, or T, can bemodified by any of numerous strategies known in the art. Sambrook etal., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. The sequence can be cleavedat appropriate sites with restriction endonuclease(s), followed byfurther enzymatic modification if desired, isolated, and ligated invitro. In the production of the gene encoding a derivative or analog ofa troponin subunit, care should be taken to ensure that the modifiedgene remains within the same translational reading frame as the troponinsubunit gene, uninterrupted by translational stop signals, in the generegion where the desired troponin activity is encoded.

Additionally, the troponin subunit encoding nucleic acid sequence can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including, butnot limited to, in vitro site-directed mutagenesis (Hutchinson et al.,1978, J. Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia), etc.

Manipulations of troponin subunit C, I, or T sequence may also be madeat the protein level. Included within the scope of the invention aretroponin subunit fragments or other fragments or analogs which aredifferentially modified during or after translation, e.g., byacetylation, phosphorylation, carboxylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage by cyanogenbromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, etc.

In addition, fragments and analogs of troponin subunits can bechemically synthesized. For example, a peptide corresponding to aportion of a troponin subunit which comprises the desired domain, orwhich mediates the desired activity in vitro, can be synthesized by useof a peptide synthesizer. Furthermore, if desired, nonclassical aminoacids or chemical amino acid analogs can be introduced as a substitutionor addition into the troponin subunit sequence. Non-classical aminoacids include, but are not limited to, the D-isomers of the common aminoacids, α-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, designer amino acids suchas β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl aminoacids.

In a specific embodiment, the invention encompasses a chimeric, orfusion, protein comprising a troponin subunit or fragment thereof(consisting of at least a domain or motif of the troponin subunit thatis responsible for inhibiting endothelial cell proliferation) joined atits amino or carboxy-terminus via a peptide bond to an amino acidsequence of a different protein. Such a chimeric product can be made byligating the appropriate nucleic acid sequences encoding the desiredamino acid sequences to each other by methods known in the art, in theproper coding frame, and expressing the chimeric product by methodscommonly known in the art. Alternatively, such a chimeric product may bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer.

ASSAYS OF TROPONIN PROTEINS FRAGMENTS AND ANALOGS

The functional activity and/or therapeutically effective dose oftroponin subunits, fragments and analogs, can be assayed in vitro byvarious methods. These methods are based on the physiological processesinvolved in angiogenesis and while they are within the scope of theinvention, they are not intended to limit the methods by which troponinsubunits, fragments and analogs inhibiting angiogenesis are definedand/or a therapeutically effective dosage of the pharmaceuticalcomposition is determined.

For example, where one is assaying for the ability of troponin subunits,fragments, and analogs, to inhibit or interfere with the proliferationof capillary endothelial cells (EC) in vitro, various bioassays known inthe art can be used, including, but not limited to, radioactiveincorporation into nucleic acids, calorimetric assays and cell counting.

Inhibition of endothelial cell proliferation may be measured bycalorimetric determination of cellular acid phosphatase activity orelectronic cell counting. These methods provide a quick and sensitivescreen for determining the number of endothelial cells in culture aftertreatment with the troponin subunit, derivative, or analog of theinvention, and an angiogenesis stimulating factor such as aFGF. Thecalorimetric determination of cellular acid phosphatase activity isdescribed by Connolly et al., 1986, J. Anal. Biochem. 152:136-140.According to this method, described in Example 3, capillary endothelialcells are treated with angiogenesis stimulating factors, such as aFGF,and a range of potential inhibitor concentrations. These samples areincubated to allow for growth, and then harvested, washed, lysed in abuffer containing a phosphatase substrate, and then incubated a secondtime. A basic solution is added to stop the reaction and colordevelopment is determined at 405 λ. According to Connolly et al., alinear relationship is obtained between acid phosphatase activity andendothelial cell number up to 10,000 cells/sample. Standard curves foracid phosphatase activity are also generated from known cell numbers inorder to confirm that the enzyme levels reflect the actual EC numbers.Percent inhibition is determined by comparing the cell number of samplesexposed to stimulus with those exposed to both stimulus and inhibitor.

Colorimetric assays to determine the effect of troponin subunits C, I,and T on endothelial cell proliferation demonstrate that all threetroponin subunits interefere with bFGF-stimulated endothelial cellproliferation.

Troponin C inhibited bFGF-stimulated endothelial cell proliferation in adose-dependent manner in all concentrations tested (FIG. 1). Percentinhibition of bovine endothelial cell proliferation ("BCE" ) was 54%,86%, 83%, and 100% at concentrations of 280 nM, 1.4 μm, 2.8 μM and 5.6μM, respectively. An inhibition of 100% was observed at a concentrationof 20 μg/well (5.6 μM). IC₅₀ represents the concentration at which 50%inhibition of aFGF growth factor-induced stimulation was observed. TheIC₅₀ of troponin C was determined to be 278 nM.

Troponin I inhibited bFGF-stimulated BCE proliferation at concentrationsof 1 and 5 μg/well, but inhibition was not observed in the sample testedat 10 μg/well (FIG. 2). The percent inhibition of BCE was 33% and 46% atconcentrations of 240 nM and 1.2 μM, respectively. The IC₅₀ of troponinI was determined to be 1.14 μM.

Troponin T inhibited bFGF-stimulated EC proliferation at concentrationsof 10 and 20 ug/well, but not at concentrations of 1 and 5 μg/well (FIG.3). BCE proliferation was inhibited 23% and 62% at 1.6 μM and 3.3 μM,respectively. The IC₅₀ of troponin T was determined to be 2.14 μM.

The combination of troponin subunits C and I inhibited EC at allconcentrations tested (FIG. 4). The percent inhibition of BCE was 52%,54% 73% and 47% at 130 nM, 645 nM, 1.3 μM and 2.6 μM, respectively. TheIC₅₀ of this combination was determined to be 110 nM.

The combination of troponin subunits C, I and T was observed to inhibitaFGF stimulated BCE proliferation by 16% at a concentration of 360 nM (5ug/well, FIG. 5).

The troponins samples tested had no detectable inhibitory effect on thegrowth of Balb/c 3T3 cells, a non-endothelial cell type.

The incorporation of radioactive thymidine by capillary endothelialcells represents another means by which to assay for the inhibition ofendothelial cell proliferation by a potential angiogenesis inhibitor.According to this method, a predetermined number of capillaryendothelial cells are grown in the presence of ³ H-Thymidine stock, anangiogenesis stimulator such as for example, bFGF, and a range ofconcentrations of the angiogenesis inhibitor to be tested. Followingincubation, the cells are harvested and the extent of thymidineincorporation is determined. See, Example 2.

The ability of varying concentrations of troponin subunits, fragments oranalogs to interfere with the process of capillary endothelial cellmigration in response to an angiogenic stimulus can be assayed using themodified Boyden chamber technique. See, Section 2 and Example 4, infra.

Another means by which to assay the functional activity of troponinsubunits, fragments and analogs, involves examining the ability of thecompounds to inhibit the directed migration of capillary endothelialcells which ultimately results in capillary tube formation. This abilitymay be assessed for example, using an assay in which capillaryendothelial cells plated on collagen gels are challenged with theinhibitor, and determining whether capillary-like tube structures areformed by the cultured endothelial cells.

Assays for the ability to inhibit angiogenesis in vivo include the chickchorioallantoic membrane assay (see Section 2 and Example 5, infra) andrat or rabbit corneal pocket assays. See, Polverini et al., 1991,Methods Enzymol. 198:440-450. According to the corneal pocket assays, atumor of choice is implanted into the cornea of the test animal in theform of a corneal pocket. The potential angiogenesis inhibitor isapplied to the corneal pocket and the corneal pocket is routinelyexamined for neovascularization. See, Section 2 and Example 6 infra.

One embodiment of the invention provides for combination of the troponinsubunits, fragments, or analogs of the present invention to inhibitangiogenesis. Another embodiment provides for the combination oftroponin subunits, fragments, or analogs with other angiogenesisinhibiting factors. Such angiogenesis inhibiting factors include, butare not limited to: angiostatic steroids, thrombospondin, plateletfactor IV, transforming growth factor β, interferons, tumor necrosisfactor α, bovine vitreous extract, protamine, tissue inhibitors ofmetalloproteinases (TIMP-1 and TIMP-2), prolactin (16-kd fragment),angiostatin (38-kd fragment of plasminogen), bfGf soluble receptor, andplacental proliferin-related protein. See, e.g., reviews by Folkman etal., 1995, N. Engl. J. Med. 333:1757-1763 and Klagsbrun et al., 1991,Annu. Rev. Physiol. 53:217-239.

The therapeutically effective dosage for inhibition of angiogenesis invivo, defined as inhibition of capillary endothelial cell proliferation,migration, and/or blood vessel ingrowth, may be extrapolated from in vitro inhibition assays using the compositions of the invention above orin combination with other angiogenesis inhibiting factors. The effectivedosage is also dependent on the method and means of delivery. Forexample, in some applications, as in the treatment of psoriasis ordiabetic retinopathy, the inhibitor is delivered in a topical-ophthalmiccarrier. In other applications, as in the treatment of solid tumors, theinhibitor is delivered by means of a biodegradable, polymeric implant.The protein can also be modified, for example, by polyethyleneglycoltreatment.

THERAPEUTIC USES

The invention provides for treatment of diseases or disorders associatedwith neovascularization by administration of a therapeutic compound ofthe invention. Such therapeutic compounds (termed herein "Therapeutics")include troponin subunits and fragments and analogs thereof (e.g., asdescribed infra).

MALIGNANCIES

Malignant and metastatic conditions which can be treated with theTherapeutic compounds of the present invention include, but are notlimited to, the solid tumors listed in Table 1 (for a review of suchdisorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. LippincottCo., Philadelphia):

                  TABLE 1                                                         ______________________________________                                        MALIGNANCIES AND RELATED DISORDERS                                                     Solid tumors                                                         ______________________________________                                                 sarcomas and carcinomas                                                       fibrosarcoma                                                                  myxosarcoma                                                                   liposarcoma                                                                   chondrosarcoma                                                                osteogenic sarcoma                                                            chordoma                                                                      angiosarcoma                                                                  endotheliosarcoma                                                             lymphangiosarcoma                                                             lymphangioendotheliosarcoma                                                   synovioma                                                                     mesothelioma                                                                  Ewing's tumor                                                                 leiomyosarcoma                                                                rhabdomyosarcoma                                                              colon carcinoma                                                               pancreatic cancer                                                             breast cancer                                                                 ovarian cancer                                                                prostate cancer                                                               squamous cell carcinoma                                                       basal cell carcinoma                                                          adenocarcinoma                                                                sweat gland carcinoma                                                         sebaceous gland carcinoma                                                     papillary carcinoma                                                           papillary adenocarcinomas                                                     cystadenocarcinoma                                                            medullary carcinoma                                                           bronchogenic carcinoma                                                        renal cell carcinoma                                                          hepatoma                                                                      bile duct carcinoma                                                           choriocarcinoma                                                               seminoma                                                                      embryonal carcinoma                                                           Wilms' tumor                                                                  cervical cancer                                                               testicular tumor                                                              lung carcinoma                                                                small cell lung carcinoma                                                     bladder carcinoma                                                             epithelial carcinoma                                                          glioma                                                                        astrocytoma                                                                   medulloblastoma                                                               craniopharyngioma                                                             ependymoma                                                                    Kaposi's sarcoma                                                              pinealoma                                                                     hemangioblastoma                                                              acoustic neuroma                                                              oligodendroglioma                                                             menangioma                                                                    melanoma                                                                      neuroblastoma                                                                 retinoblastoma                                                       ______________________________________                                    

OCULAR DISORDERS

Ocular disorders associated with neovascularization which can be treatedwith the Therapeutic compounds of the present invention include, but arenot limited to:

neovascular glaucoma

diabetic retinopathy

retinoblastoma

retrolental fibroplasia

uveitis

retinopathy of prematurity

macular degeneration

corneal graft neovascularization

as well as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., 1978, Am. J. Ophthal. 85:704-710 and Gartner et al.,1978, Surv. Ophthal. 22:291-312.

OTHER DISORDERS

Other disorders which can be treated with the Therapeutic compounds ofthe present invention include, but are not limited to, hemangioma,arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayedwound healing, granulations, hemophilic joints, hypertrophic scars,nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY

The Therapeutics of the invention can be tested in vivo for the desiredtherapeutic or prophylactic activity as well as for determination oftherapeutically effective dosage. For example, such compounds can betested in suitable animal model systems prior to testing in humans,including, but not limited to, rats, mice, chicken, cows, monkeys,rabbits, etc. For in vivo testing, prior to administration to humans,any animal model system known in the art may be used.

THERAPEUTIC/PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS

The invention provides methods of treatment (and prophylaxis) byadministration to a subject an effective amount of a Therapeutic of theinvention. In a preferred aspect, the Therapeutic is substantiallypurified as set forth in Example 1. The subject is preferably an animal,including, but not limited to, animals such as cows, pigs, chickens,etc., and is preferably a mammal, and most preferably human.

The invention further provides methods of treatment by administration toa subject, an effective amount of a Therapeutic of the inventioncombined with a chemotherapeutic agent and/or radioactive isotopeexposure.

The invention also provides for methods of treatment of a Therapeutic ofthe invention for patients who have entered a remission in order tomaintain a dormant state.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis (see, e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. It is preferred thatadministration is localized, but it may be systemic. In addition, it maybe desirable to introduce the pharmaceutical compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

For topical application, the purified troponin subunit is combined witha carrier so that an effective dosage is delivered, based on the desiredactivity (i.e., ranging from an effective dosage, for example, of 1.0 μMto 1.0 mM to prevent localized angiogenesis, endothelial cell migration,and/or inhibition of capillary endothelial cell proliferation. In oneembodiment, a topical troponin subunit, fragment or analog is applied tothe skin for treatment of diseases such as psoriasis. The carrier may inthe form of, for example, and not by way of limitation, an ointment,cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.

A topical Therapeutic for treatment of some of the eye disordersdiscussed infra consists of an effective amount of troponin subunit,fragment, or analog, in a ophthalmologically acceptable excipient suchas buffered saline, mineral oil, vegetable oils such as corn or arachisoil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes orliposome-like products. Any of these compositions may also includepreservatives, antioxidants, antibiotics, immunosuppressants, and otherbiologically or pharmaceutically effective agents which do not exert adetrimental effect on the troponin subunit.

For directed internal topical applications, for example for treatment ofulcers or hemorrhoids, the troponin subunit, fragment, or analogcomposition may be in the form of tablets or capsules, which can containany of the following ingredients, or compounds of a similar nature: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; or a glidant such as colloidal silicon dioxide.When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar, shellac, or other enteric agents.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome. See, Langer et al., 1990, Science 249:1527-1533;Treat et al., 1989, in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353 14365; Lopez-Berestein, ibid., pp. 317-327.

In yet another embodiment, the Therapeutic can be delivered in acontrolled release system. In one embodiment, an infusion pump may beused to administer troponin subunit, such as for example, that used fordelivering insulin or chemotherapy to specific organs or tumors (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed., 1987, Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574.

In a preferred form, the troponin subunit, fragment, or analog isadministered in combination with a biodegradable, biocompatiblepolymeric implant which releases the troponin subunit, fragment, oranalog over a controlled period of time at a selected site. Examples ofpreferred polymeric materials include polyanhydrides, polyorthoesters,polyglycolic acid, polylactic acid, polyethylene vinyl acetate, andcopolymers and blends thereof. See, Medical Applications of ControlledRelease, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.;Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), 1984, Wiley, N.Y.; Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 71:105. In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,1989, supra, vol. 2, pp. 115-138).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

In a specific embodiment, the term "pharmaceutically acceptable" meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm "carrier" refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents. Water is apreferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents such as acetates,citrates or phosphates. Antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose are alsoenvisioned. The parental preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides, microcrystalline cellulose, gum tragacanth or gelatin.Oral formulation can include standard carriers such as pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate, etc. Examples of suitablepharmaceutical carriers are described in "Remington's PharmaceuticalSciences" by E. W. Martin. Such compositions will contain atherapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays such as thosediscussed in section 5.2 may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.However, suitable dosage ranges for intravenous administration aregenerally about 20-500 micrograms of active compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test bioassays or systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Modifications and variations of the compositions of the presentinvention, and methods for use, will be obvious to those skilled in theart from the foregoing detailed description. Such modifications andvariations are intended to fall within the scope of the appended claims.

The following non-limiting examples demonstrate the discovery oftroponin subunit inhibition of angiogenic stimulus induced endothelialcell proliferation, and means for determining the effective dosage oftroponin subunit, fragment, or analog to inhibit angiogenesis, as wellas for identifying troponin subunit fragments and analogs (i.e., thosefragments or analogs of troponin subunit capable of inhibitingangiogenesis. The troponin subunit used in the examples is purified asdescribed infra.

EXAMPLES

Example 1: Purification of Troponin subunit Components

Cardiac Troponin Isolation from Tissue

The procedures of Ebashi et al., 1968, J. Biochem. 64:465-477; Yasui etal., 1968, J. Biol. Chem. 243:735-742; Hartshorne et al., 1969, Biochim.Biophys. Acta, 175:30; Schaub et al., 1969, Biochem. J. 115:993-1004;Greaser et al., 1971, J. Biol. Chem. 246:4226-4233; and Greaser et al.,1973, J. Biol. Chem. 248:2125-2133 for purifying troponin can be used.Rabbit back and leg muscles are removed, cleaned of fat and connectivetissue, and ground. The ground muscle (1 kg) is stirred for 5 min. in 2liters of a solution containing 20 mM KCl, 1 mM KHCO₃, 0.1 mM CaCl₂, and0.1 mM DTT.¹ The suspension is filtered through cheesecloth, and thewashing of the residue is repeated four times. Two liters of 95% ethanolare then added to the washed residue and the solution filtered after 10min. The ethanol extraction is repeated twice. The residue is thenwashed 3 times with 2 liters of diethyl ether for 10 min. Finally theresidue is allowed to dry at room temperature for 2 to 3 hours.

The dried powder (from 1 kg of muscle) is extracted overnight at 22°with 2 liters of a solution containing 1M KCl, 25 mM Tris (pH 8.0), 0.1mM CaCl², and 1 mM DTT. After filtration through cheesecloth, theresidue is once more extracted with 1 liter of 1M KCl.

The extracts are combined and cooled to 4° C. Solid ammonium sulfate isadded to produce approximately 40% saturation (230 g per liter). After30 min. the solution is centrifuged and 125 g of ammonium sulfate isthen added per liter of supernatant (60% saturation). Aftercentrifugation the precipitate is dissolved in 500 ml of a solutioncontaining 5 mM Tris (pH 7.5), 0.1 mM CaCl₂, and 0.1 mM DTT and dialyzedagainst 15 liters of the same solution for 6 hours and against a freshsolution overnight.

Solid KCl is added to a final concentration of 1M and 1M KCl solution isadded to bring the volume to 1 liter. The pH is then adjusted to 4.6 byaddition of HCl, and the tropomyosin precipitate is removed bycentrifugation. The pH of the supernatant is adjusted to 7.0 with KOH,and 450 g of ammonium sulfate were added per liter (70% saturation). Theprecipitate is dissolved in a solution containing 5 mM Tris (pH 7.5, 0.1mM CaCl₂, and 0.1 mM DTT, and dialyzed overnight against the samesolution. Solid KCl is added to bring its concentration to 1M, the pHadjusted to 4.6, and the precipitate formed removed by centrifugation.The neutralized supernatant is dialyzed against 2 mM Tris (pH 7.5) untilthe Nessler reaction is negative. The final yield of troponin is usually2.5 to 3.0 g per kg of fresh muscle.

Cardiac Troponin Isolation from Tissue

Bovine hearts are obtained approximately 30 min. after death andimmediately cut open, rinsed of blood, and immersed in ice. The leftventricle is removed, trimmed of excess fat and connective tissue, andground. All subsequent extraction and preparation steps are performed at0°-3° except where noted. The ground muscle (500 g) is homogenized in aWaring Blender for 1 min. in 2.5 liters of solution containing 0.09M KH₂PO₄, 0.06M K₂ HPO₄, 0.3M KCl, 5 mM 2-mercaptoethanol, pH 6.8. Thehomogenized muscle suspension is then stirred for 30 min. andcentrifuged at 1000 ×g for 20 min. The precipitate is re-extracted for30 min. and centrifuged. The residue is then washed with 2.5 liters of 5mM 2-mercaptoethanol and centrifuged at 1000 ×g for 10 min., followed bytwo successive washings and centrifugations with 1.5 liters of 50 mMKCl, 5 mM Tris-HCl (pH 8.1/5), mM 2-mercaptoethanol. The residue is thenwashed and centrifuged twice with 1.5 liters of 50 mM Tris-HCl (pH 8.1),and 5 mM 2-mercaptoethanol. The volume of the residue is measured, andthe residue is mixed with 0.5 volume of 3 M KCl, 50 mM Tris-HCl (pH8.1), and 5 mM 2-mercaptoethanol. After a 16- to 20-hour extraction at0°, the suspension is centrifuged at 15,000 ×g for 10 min. The sedimentis discarded, and the supernatant is adjusted to pH 7.6 with 0.05 N HCl.The filamentous precipitate which forms upon pH adjustment is removed byfiltering the extract through nylon gauze. The protein that precipitatesbetween 30 and 50% ammonium sulfate saturation is collected, dissolvedin a solution containing 1M KCl, and 1 mM potassium phosphate (pH 6.8),and 5 mM 2-mercaptoethanol, and dialyzed against the same solution for 4hours and against a fresh solution overnight. The protein solution isclarified by centrifugation at 105,000 ×g for 30 min. The troponin isthen purified by chromatography on a hydroxylapatite column with theprotein being eluted between 0.08 and 0.10M phosphate. Greaser et al.,1972 Cold Spring Harbor Symp. Quant. Biol. 37:235-244. Rabbit cardiactroponin is prepared in a similar manner using a pooled batch of heartswhich has been stored at -20° C. prior to extraction.

The troponin subunits are separated by DEAE-Sephadex chromatography in6M urea. Bovine cardiac tropomyosin is prepared from the 50% ammoniumsulfate saturation supernatant from the troponin extraction scheme (seeabove). Ammonium sulfate is added to 65% saturation, and the precipitateis dissolved in and dialyzed versus 1M KCl, 1 mM potassium phosphate (pH7.0), and 5 mM 2-mercaptoethanol. The protein is then purified byhydroxylapatite chromatography.

Protein Determination--Protein concentrations are determined by thebiuret method of Gornall et al. using bovine serum albumin as astandard. Gornall et al., 1949, J. Biol. Chem., 177:751-766.

Separation of Components--A sequence of SP-Sephadex and DEAE-Sephadexchromatography gives complete separation of the three cardiac troponincomponents.

Recombinant Troponin Isolation and Reconstitution Protocols Troponin Iand T

DNA encoding various troponin subunits and isoforms are known in theart. See, e.g., Wu et al., 1994, DNA Cell. Biol. 13:217-233; Schreier etal., 1990, J. Biol. Chem. 265:21247-21253; and Gahlmann et al., 1990, J.Biol. Chem. 265:12520-12528.

To express a troponin subunit, DNA encoding the subunit is subclonedinto a high copy number expression plasmid, such as KP3998, usingrecombinant techniques known in the art.

To express the cloned cDNA, E. coli transformed with theinsert-containing pKP1500 vector is grown overnight at 37° C., theninoculated into 4 liters of Luria-Bertani broth (LB) medium and grown at42° C. until mid-log phase. Isopropyl-1-thio-β-D-galactopyranoside isthen added to 0.5 mM, and the culture is allowed to grow at 42° C.overnight. Purification of expressed troponin subunit, fragment, oranalog may be adapted from published procedures (Reinach et al., 1988,J. Biol. Chem. 250:4628-4633 and Xu et al., 1988, J. Biol. Chem.263:13962-13969). The cells are harvested by centrifugation andsuspended in 20 ml of 20 mM Tris, 20% sucrose, 1 mM EDTA, 0.2 mMphenylmethylsulfonyl fluoride, 1 mg/ml lysozyme, pH 7.5. Afterincubation on ice for 30 min., 80 ml of 20 mM Tris, 1 mM EDTA, 0.2 mMphenylmethylsulfonyl fluoride, 0.5 mM DTT is added and the cells brokenin a French press (SLM Instruments). The cell debris is pelleted; thesupernatant is made 35% in saturated (NH₄)₂ SO₄ and stirred on ice for30 min. After sedimentation, the supernatant is made 50 mM in NaCl, 5 mMin CaCl₂, 1 mM in MgCl₂, and 1 mM in DTT and then loaded onto a 1.5×25-cm phenyl-Sepharose (Pharmacia LKB Biotechnology Inc.) column. Thecolumn is washed first with 50 mM Tris, 50 mM NaCl, 5 mM CaCl₂, 1 mMMgCl₂, 1 mM DTT, pH 7.5, then with 50 mM Tris, 1 mM NaCl, 0.1 mM CaCl₂,1 mM DTT, pH 7.5, until no more protein is eluted. The crude troponinsubunit is then eluted with 50 mM Tris, 1 mM EDTA, 1 mM DTT, pH 7.5.Fractions that contained troponin subunit, fragment, or analog arepooled, dialyzed against 25 mM Tris, 6M urea (United States BiochemicalCorp.), 1 mM MgCl₂, 1 mM DTT, pH 8.0, and loaded onto a 1.5×25-cm DE52(Whatman) column. The column is eluted with a 0-0.6M NaCl lineargradient. Troponin subunit, fragment, or analog eluted from the columnis dialyzed against 0.1 mM NH₄ HCO₃, 1 mM β-mercaptoethanol,lyophilized, and stored. Purity is assessed by SDS-polyacrylamide gelelectrophoresis and UV spectrophotometry. Typical yields of 6 mg ofpurified recombinant troponin subunit, fragment, or analog/liter ofbacterial culture are expected.

The lyophilized recombinant protein is resuspended in a take up bufferconsisting of 6M urea, 20 mM Hepes (pH 7.5), 0.5M NaCl, 2 mM EDTA, and 5mM DTT. The mixture is nutated at room temperature for 1 hour. Thesolution is then dialyzed at 4° C. for six hours with 1 exchange againsta dialysis buffer consisting of 0.5M NaCl, 20 mM Hepes (pH 7.5), and 0.5mM DTT.

Protein concentration is determined for each subunit at 280λ. Theextension coefficient of Troponin I is 0.40 and Troponin T is 0.50.

Troponin C

The lyophilized recombinant protein is resuspended in a take up bufferconsisting of 0.1M NaCl, 20 mM Hepes (pH 7.5), 2 mM EDTA, and 5 mM DTT.This solution is dialyzed for 6 hours at 4° C. with one exchange againsta dialysis buffer of 0.1M NaCl, 20 mM Hepes (pH 7.5), and 0.5 mM DTT.

Protein concentration is determined by measuring absorbance at 280λ. Theextension coefficient for troponin C is 0.18.

Reconstitution of Combined Subunits:

Protein concentrations having the same reconstitution molar ratios oftroponin subunits C, I, and T are maintained for all variouscombinations. These concentrations of the respective proteins arecombined in a reconstitution buffer consisting of 0.1M NaCl, 0.1M CaCl2,mM DTT, 5 mM Hepes (pH 7.5). Dialysis is for 20-24 hours at 4° C. withthree exchanges over a dialysis buffer consisting of 0.1M NaCl, 0.1 mCaCl₂, 0.5 mM DTT, and 5 mM Hepes (pH 7.5).

Protein concentration is approximated by measuring absorption at 278λ.The troponin trimer has an extension coefficient of 0.45 at 278λ.

Example 2: Inhibition of Endothelial Cell Proliferation measured by DNAsynthesis.

The inhibitory effect of troponin subunit, fragment, or analog on theproliferation of bFGF-stimulated EC can be measured according to thefollowing procedure.

Endothelial cell DNA Synthesis:

On day one, 5,000 Bovine capillary endothelial cells in DMEM/10% CS/1%GPS are plated onto each well of a 96-well pregelatinized tissue cultureplate. On day two, the cell media is changed to DMEM, 2% CS, 1% GPS,0.5% BSA (complete medium), supplemented with 10 μl of 1 mg/ml "cold"thymidine per 50 ml of medium. On day three, test samples in completemedium are added in duplicate. Additionally, bFGF is added in each wellexcept for the appropriate controls, to a final concentration of 0.2ng/well. On day four, 5 μl of 1:13 diluted ³ H-Thymidine stock is addedto each well and the plate is incubated for 5-6 hours. Followingincubation, the medium is aspirated, and the remainder is rinsed oncewith PBS, then twice for 5 minutes each with methanol followed by tworinses each for 10 minutes with 5% TCA. The cells well contents are thenrinsed with water three times, dried to the plate, and 100 μl of 0.3 NNaOH is added to each well. The contents of the well are then transferedto the scintillation counter vials and 3 mls of Ecolume added to eachvial. Samples are then counted on the scintillation counter.

3T3 Cell DNA Synthesis:

DNA synthesis in bFGF-stimulated 3T3 cells provides a control with whichto evaluate results obtained for bFGF stimulated endothelial cellproliferation. DNA synthesis in the 3T3 cells can be determinedaccording to the following method.

BALB/c 3T3 cells are trypsinized and resuspended at a concentration of5×10⁴ cells/ml. Aliquots of 200 μl are plated into 0.3 cm² microtiterwells (Microtest II tissue Culture Plates, Falcon). After reachingconfluence, in a period of 2 to 3 days, the cells are further incubatedfor a minimum of 5 days in order to deplete the media of growthpromoting factors. These growth conditions yield confluent monolayers ofnon-dividing BALB/c 3T3 cells. Test samples are dissolved in 50 μl of0.15M NaCl and added to microtiter wells, along with ³ H!TdR. After anincubation of at least 24 hours, the media is removed and the cells arewashed in PBS. Fixation of the cells and removal of unincorporated ³H!TdR is accomplished by the following successive steps; addition ofmethanol twice for periods of 5 minutes, 4 washes with H₂ O, addition ofcold 5% TCA twice for periods of 10 minutes, and 4 washes with H₂ O. DNAsynthesis is measured either by liquid scintillation counting or byautoradiography using a modification of the method described byHaudenschild et al., 1976, M. Exp. Cell Res. 98:175. For scintillationcounting, cells are lysed in 150 μl of 0.3 N NaOH and counted in 5 ml.of Insta-Gel liquid scintillation cocktail (Packard) using a PackardTri-Carb liquid scintillation counter. Alternatively, autoradiographymay be used to quantitate DNA synthesis by punching out the bottoms ofthe microtiter wells and mounting them on glass slides with silasticglue. The slides are dipped in a 1 g/ml solution of NTB2 nuclear trackemulsion (Kodak) and exposed for 3-4 days. The emulsion is developedwith Microdol-X solution (Kodak) for 10 minutes, rinsed with distilledH₂ O, and fixed with Rapid Fixer (Kodak) for three minutes. Theautoradiographs are stained with a modified Giemsa stain. At least 1000nuclei are counted in each well and DNA synthesis, expressed as thepercentage of nuclei labeled. Cell division is measured by counting thenumber of cells in microtiter wells with the aid of a grid after 40-48hour incubations with test samples.

Example 3: Inhibition of Endothelial Cell Proliferation measured bycalorimetric determination of cellular acid phosphatase activity andelectronic cell counting

A quick and sensitive screen for inhibition of EC proliferation inresponse to treatment with a troponin subunit, analog, or derivative ofthe invention involves incubating the cells in the presence of varyingconcentrations of the inhibitor and determining the number ofendothelial cells in culture based on the calorimetric determination ofcellular acid phosphatase activity, described by Connolly, et al., 1986,J. Anal. Biochem. 152:136-140.

We measured the effect of troponin on the proliferation of capillaryendothelial cells (EC) in an assay which measures the ability of thisprotein to interfere with stimulation of endothelial cell proliferationby a known angiogenesis factor (bFGF).

Capillary endothelial cells and Balb/c 3T3 cells were separately plated(2 ×10³ /0.2 ml) onto gelatin-coated 96-well tissue culture dishes onday 1. On day 2, cells were refed with Dulbecco's modified Eagle'smedium (Gibco) with 5% calf serum (Hyclone) (DMEM/5) and bFGF (10 ng/ml)(FGF Co.) and increasing concentrations of the troponin subunit. Thesesubstances were added simultaneously in volumes that did not exceed 10%of the final volume. Wells containing phosphate buffered saline (PBS)(Gibco) alone and PBS+bFGF were included as controls. On day 5, mediawas removed and cells were washed with PBS and lysed in 100 μl of buffercontaining 0.1M sodium acetate (pH 5.5), 0.1% Triton X-100™ and 100 mMp-nitrophenyl phosphate (Sigma 104 phosphatase substrate). Afterincubation for 2 hours at 37° C., the reaction was stopped with theaddition of 10 μl of 1 N NAOH. Color development was determined at 405nm using a rapid microplate reader (Bio-Tek).

Percent inhibition was determined by comparing the cell number of wellsexposed to stimulus with those exposed to stimulus and troponinsubunits.

All three troponin subunits were found to inhibit bFGF-stimulated ECproliferation, as measured by the calorimetric assay.

Troponin C inhibited bFGF-stimulated endothelial cell proliferation in adose-dependent manner in all concentrations tested (FIG. 1). Percentinhibition of bovine endothelial cell proliferation ("BCE") was 54%,86%, 83%, and 100% at concentrations of 280 nM, 1.4 μM, 2.8 μM and 5.6μM, respectively. An inhibition of 100% was observed at a concentrationof 20 ug/well (5.6 μM). IC₅₀ represents the concentration at which 50%inhibition of bFGF growth factor-induced stimulation was observed. TheIC₅₀ of troponin C was determined to be 278 nM.

Troponin I inhibited bFGF-stimulated BCE proliferation at concentrationsof 1 and 5 ug/well, but inhibition was not observed in the sample testedat 10 ug/well (FIG. 2). The percent inhibition of BCE was 33% and 46% atconcentrations of 240 nM and 1.2 μM, respectively. The IC₅₀ of troponinI was determined to be 1.14 μM.

Troponin T inhibited bFGF-stimulated EC proliferation at concentrationsof 10 and 20 ug/well, but not at concentrations of 1 and 5 μg/well (FIG.3). BCE proliferation was inhibited 23% and 62% at 1.6 μM and 3.3 μM,respectively. The IC₅₀. of troponin T was determined to be 2.14 μM.

The combination of troponin subunits C and I inhibited EC at allconcentrations tested (FIG. 4). The percent inhibition of proliferationof BCE was 52%, 540% 73% and 47% at 130 nM, 645 nM, 1.3 μM and 2.6 μM,respectively. The IC₅₀ of this combination was determined to be 110 nM.

The combination of troponin subunits C, I and T was observed to inhibitbFGF-stimulated BCE proliferation by 16% at a concentration of 360 nM (5ug/well, FIG. 5).

The troponin samples tested had no detectable inhibitory effect on thegrowth of Balb/c 3T3 cells, a non-endothelial cell type.

Example 4: Inhibition of Capillary Endothelial Cell Migration bytrotonin

Determination of the ability of the troponin subunit, derivative, oranalog to inhibit the angiogenic process of capillary EC migration inresponse to an angiogenic stimulus, can be determined using amodification of the Boyden chamber technique is used to study the effectof troponin subunit, derivative, or analog on capillary EC migration.Falk et al., 1980, J. Immunol. 118:239-247 (1980). A blind-well Boydenchamber, consists of two wells (upper and lower) separated by a porousmembrane. J. Exp. Med. 115:453-456 (1962). A known concentration ofgrowth factor is placed in the lower wells and a predetermined number ofcells and troponin subunit, derivative, or analog is placed in the upperwells. Cells attach to the upper surface of the membrane, migratethrough and attach to the lower membrane surface. The membrane can thenbe fixed and stained for counting, using the method of Glaser et al.,1980, Nature 288:483-484.

Migration is measured using blind well chambers (Neuroprobe, no.025-187) and polycarbonate membranes with 8 micron pores (Nucleopore)precoated with fibronectin (6.67 μg/ml in PBS) (human, Cooper). BasicFGF (Takeda Co.) diluted in DMEM with 1% calf serum (DMEM/1) is added tothe lower well at a concentration of 10 ng/ml. The upper wells receive5×10⁵ capillary EC/ml and increasing concentrations of purified troponinsubunit, derivative or analog is used within 24 hours of purification.Control wells receive DMEM/1, either with or without bFGF. The migrationchambers are incubated at 37° C. in 10% CO2 for 4 hours. The cells onthe upper surface of the membrane are then wiped off by drawing themembrane over a wiper blade (Neuroprobe). The cells which have migratedthrough the membrane onto the lower surface are fixed in 2%glutaraldehyde followed by methanol (4° C.) and stained withhematoxylin. Migration is quantified by counting the number of cells onthe lower surface in 16 oil immersion fields and comparing this numberwith that obtained for the control.

Example 5: Inhibition in vivo of Neovascularization by troponin asdetermined by the chick chorioallantoic membrane assay

The chick chorioallantoic membrane assay (CAM), may be used to determinewhether troponin subunit, derivative or analog is capable of inhibitingneovascularization in vivo. Taylor and Folkman, 1982, Nature (London)297:307-312. The effect of troponin subunit, derivative or analog ongrowing embryonic vessels is studied using chick embryos in whichcapillaries appear in the yolk sac at 48 h and grow rapidly over thenext 6-8 days.

Three day post fertilization chick embryos are removed from their shellsand placed in plastic petri dishes (1005, Falcon). The specimens aremaintained in humidified 5% CO₂ at 37° C. On day 6 of development,samples of purified troponin subunit, derivative or analog are mixed inmethylcellulose disks and applied to the surfaces of the growing CAMsabove the dense subectodermal plexus. Control specimens in which CAMsare implanted with empty methylcellulose disks are also prepared. TheCAMs are injected intravascularly with India ink/Liposyn to more clearlydelineate CAM vascularity. Taylor et al., 1982, Nature 297:307-312.

Following a 48 hour exposure of the CAMs to the troponin subunit,derivative, or analog, the area around the implant is observed andevaluated. Test specimens having avascular zones completely free ofIndia-ink filled capillaries surrounding the test implant indicate thepresence of an inhibitor of embryonic neovascularization. In contrast,the control specimens show neovascularization in close proximity or incontact with the methylcellulose disks.

Histological mesodermal studies are preformed on the CAMs of test andcontrol specimens. The specimens are embedded in JB-4 plastic(Polysciences) at 4° C. and 3 μm sections are cut using a Reichert 2050microtome. Sections are stained with toluidine blue and micrographs aretaken on a Zeiss photomicroscope using Kodak TM x100 and a green filter.

Example 6: Inhibition in vivo of Neovascularization by troponin asdetermined by the rabbit corneal Pocket assay

Male NZW rabbits weighing 4-5 lbs. are anesthetized with intravenouspentobarbital (25 mg/kg) and 2% xylocaine solution is applied to thecornea. The eye is proptosed and rinsed intermittently with Ringer'ssolution to prevent drying. The adult rabbit cornea has a diameter ofapproximately 12 mm. An intracorneal pocket is made by an incisionapproximately 0.15 mm deep and 1.5 mm long in the center of the corneawith a No. 11 scalpel blade, using aseptic technique. A 5 mm-long pocketis formed within the corneal stroma by inserting a 1.5 mm wide,malleable iris spatula. In the majority of animals, the end of thecorneal pocket is extended to within 1 mm of the corneal-scleraljunction. In a smaller series of 22 rabbits implanted with tumor alone,pockets are placed at greater distances--2-6 mm from the corneal-scleraljunction by starting the incision away from the center.

In the first assay, polymer pellets of ethylene vinyl acetate (EVAc)copolymer are impregnated with test substance and surgically implantedin a pocket in the rabbit cornea approximately 1 mm from the limbus.When this assay system is being used to test for angiogenesisinhibitors, either a piece of V2 carcinoma or some other angiogenicstimulant is implanted distal to the polymer, 2 mm from the limbus. Onthe opposite eye of each rabbit, control polymer pellets that are emptyare implanted next to an angiogenic stimulant in the same way. In thesecontrol corneas, capillary blood vessels start growing towards the tumorimplant in 5-6 days, eventually sweeping over the blank polymer. In testcorneas, the directional growth of new capillaries from the limbal bloodvessels towards the tumor occurs at a reduced rate and is ofteninhibited such that an avascular region around the polymer is observed(FIG. 1). This assay is quantitated by measurement of the maximum vessellengths with a stereoscopic microscope.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 160 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: None                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..160                                                          (D) OTHER INFORMATION: /label=Human Fast Twitch Skeletal                      Muscle Troponin C                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetThrAspGlnGlnAlaGluAlaArgSerTyrLeuSerGluGluMet                              151015                                                                        IleAlaGluPheLysAlaAlaPheAspMetPheAspAlaAspGlyGly                              202530                                                                        GlyAspIleSerValLysGluLeuGlyThrValMetArgMetLeuGly                              354045                                                                        GlnThrProThrLysGluGluLeuAspAlaIleIleGluGluValAsp                              505560                                                                        GluAspGlySerGlyThrIleAspPheGluGluPheLeuValMetMet                              65707580                                                                      ValArgGlnMetLysGluAspAlaLysGlyLysSerGluGluGluLeu                              859095                                                                        AlaGluCysPheArgIlePheAspArgAsnAlaAspGlyTyrIleAsp                              100105110                                                                     ProGluGluLeuAlaGluIlePheArgAlaSerGlyGluHisValThr                              115120125                                                                     AspGluGluIleGluSerLeuMetLysAspGlyAspLysAsnAsnAsp                              130135140                                                                     GlyArgIleAspPheAspGluPheLeuLysMetMetGluGlyValGln                              145150155160                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: None                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..182                                                          (D) OTHER INFORMATION: /label=Human Fast Twitch Skeletal                      Muscle Troponin I                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyAspGluGluLysArgAsnArgAlaIleThrAlaArgArgGln                              151015                                                                        HisLeuLysSerValMetLeuGlnIleAlaAlaThrGluLeuGluLys                              202530                                                                        GluGluSerArgArgGluAlaGluLysGlnAsnTyrLeuAlaGluHis                              354045                                                                        CysProProLeuHisIleProGlySerMetSerGluValGlnGluLeu                              505560                                                                        CysLysGlnLeuHisAlaLysIleAspAlaAlaGluGluGluLysTyr                              65707580                                                                      AspMetGluValArgValGlnLysThrSerLysGluLeuGluAspMet                              859095                                                                        AsnGlnLysLeuPheAspLeuArgGlyLysPheLysArgProProLeu                              100105110                                                                     ArgArgValArgMetSerAlaAspAlaMetLeuLysAlaLeuLeuGly                              115120125                                                                     SerLysHisLysValCysMetAspLeuArgAlaAsnLeuLysGlnVal                              130135140                                                                     LysLysGluAspThrGluLysGluArgAspLeuArgAspValGlyAsp                              145150155160                                                                  TrpArgLysAsnIleGluGluLysSerGlyMetGluGlyArgLysLys                              165170175                                                                     MetPheGluSerGluSer                                                            180                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 258 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: None                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..258                                                          (D) OTHER INFORMATION: /label=Human Fast Skeletal Beta                        Troponin T                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetSerAspGluGluValGluGlnValGluGluGlnTyrGluGluGlu                              151015                                                                        GluGluAlaGlnGluGluGluGluValGlnGluAspThrAlaGluGlu                              202530                                                                        AspAlaGluGluGluLysProArgProLysLeuThrAlaProLysIle                              354045                                                                        ProGluGlyGluLysValAspPheAspAspIleGlnLysLysArgGln                              505560                                                                        AsnLysAspLeuMetGluLeuGlnAlaLeuIleAspSerHisPheGlu                              65707580                                                                      AlaArgLysLysGluGluGluGluLeuValAlaLeuLysGluArgIle                              859095                                                                        GluLysArgArgAlaGluArgAlaGluGlnGlnArgIleArgAlaGlu                              100105110                                                                     LysGluArgGluArgGlnAsnArgLeuAlaGluGluLysAlaArgArg                              115120125                                                                     GluGluGluAspAlaLysArgArgAlaGluAspAspLeuLysLysLys                              130135140                                                                     LysAlaLeuSerSerMetGlyAlaAsnTyrSerSerTyrLeuAlaLys                              145150155160                                                                  AlaAspGlnLysArgGlyLysLysGlnThrAlaArgGluMetLysLys                              165170175                                                                     LysIleLeuAlaGluArgArgLysProLeuAsnIleAspHisLeuGly                              180185190                                                                     GluAspLysLeuArgAspLysAlaLysGluLeuTrpGluThrLeuHis                              195200205                                                                     GlnLeuGluIleAspLysPheGluPheGlyGluLysLeuLysArgGln                              210215220                                                                     LysTyrAspIleThrThrLeuArgSerArgIleAspGlnAlaGlnLys                              225230235240                                                                  HisSerLysLysAlaGlyThrProAlaLysGlyLysValGlyGlyArg                              245250255                                                                     TrpLys                                                                        __________________________________________________________________________

We claim:
 1. A method of inhibiting atopic angiogenesis in a subject,having a disease or disorder causing atopic angiogenesis comprisingapplying to a site of atopic angiogenesis an amount of a peptide that iseffective to inhibit angiogenesis, in which the peptide is:a. aninhibitor of bFGF-stimulated bovine endothelial cell proliferationhaving an IC₅₀ of at least 10 μM; b. greater than 75 amino acids inlength; and c. greater than 80% homologous with a subunit selected fromthe group consisting of human fast-twitch troponin I subunit (SEQ. ID.NO:2), human fast-twitch troponin C subunit (SEQ. ID. NO:1) and humanfast-twitch troponin T subunit (SEQ. ID. NO:3).
 2. The method of claim1, in which the subunit is human fast-twitch troponin C (SEQ. ID. NO:1)or human fast-twitch troponin I.
 3. The method of claim 2, in which thedisease disorder is a solid tumor.
 4. The method of claim 2, in whichthe tumor is a tumor of the central nervous system.
 5. The methodaccording to claim 2, in which the disease or disorder is anophthalmologic disease or disorder.